One of the most important capabilities that enables the characterization and preparation of bio-materials throughout the life sciences is the recognition of target components in a mixture and the ability to selectively manipulate, interact, and/or isolate them. Methods known in the art to accomplish these steps include magnetic labeling techniques. In these methods, magnetically-susceptible particles (herein termed “magnetic labels” or “labels”) are used that can be attracted to a magnet and have modified surfaces that bind preferentially to target particles, cells, or molecules (herein termed “target analytes”). The surface characteristics of the labels that provide for preferential binding can include, but are not restricted to, antibodies, chemically-reactive groups, and receptor ligands. Such surface-modified magnetically-susceptible labels tend to become attached to target analytes to which they have preferential binding capacities within a mixture of analytes.
After attachment, the magnetic labels may be collected by an inhomogeneous magnetic field created by a magnet that is usually equipped with a mechanism for increasing the magnetic field inhomogeneity in the vicinity of the labels. Analytes in the mixture having negligible magnetic susceptibility and that have not become bound to the magnetically susceptibility labels to form analyte-label complexes are not collected by the magnet and can be washed away. Subsequently, the magnetic field can be removed and the analyte-label complexes can be released and collected in a separate fraction.
Thus, by using these differential trapping characteristics, current magnetic labeling methods allow target analytes to be isolated from a mixture of particles, cells, or molecules. These magnetic labeling methods can be used to retain the target analytes for further processing, analysis, or study (known in the art as “positive selection”). Alternatively, the analytes that are not retained by the magnetic field may be collected and used for further processing, analysis, or study (known in the art as “negative selection”). While the use of magnetic labeling methods is widespread, current methods have a number of significant disadvantages. For example, because all magnetic labels in a mixture are attracted to the collection magnet regardless of any differences there may be in their surface modification or binding state with target analytes, it is impossible to discriminate between, and isolate, multiple target analytes simultaneously. It is also impossible to determine the extent to which magnetic labels have bound a target analyte without additional measurement steps after magnetic collection. For example, it is impossible to distinguish between or isolate cell subpopulations that are characterized by variations in the number of labels bound to their surfaces since all cells that bind labels, regardless of the number, are collected by current magnetic methods. Finally, current methods depend on trapping the magnetic labels on surfaces in a collection chamber or column and the target analytes tend to be collected in clumps. This typically limits sample recovery because of adhesion to the chamber or column and may entrap unwanted unlabelled analytes within the labeled analytes thereby limiting the purity of the recovered target analytes.
A newer approach to the discrimination, manipulation, separation and isolation of target analytes from a mixture is based on the exploitation of the dielectric properties of the target analytes themselves or the use of dielectric labeling techniques. In U.S. Pat. Nos. 5,993,630 and 5,888,370 which are hereby expressly incorporated herein by reference, certain of the inventors of the present application teach the use of dielectrophoretic methods for the discrimination, separation and isolation of particles by exploiting their intrinsic dielectric properties in conjunction with the characteristics of a hydrodynamic flow profile.
In a concurrently filed provisional patent application concerning dielectric beads for the identification and sorting of target agents, the inventors teach methods by which labels that incorporate useful dielectric and magnetic properties may be designed. Such labels allow target analytes to be discriminated and manipulated by dielectrophoretic methods. By combining magnetic and dielectric properties as useful attributes of the labels, those methods allow for additional levels of discrimination between both the labels themselves and analyte-label complexes. For example, the disclosure teaches how different types of labels may be designed that have distinct “dielectric fingerprints” that allow for the recognition of the different label types within a “cocktail” of different label types. Because analytes, labels or analyte-label complexes do not have to be trapped in a column in order to achieve separations in these dielectric methods, they are less susceptible to entrapping unlabeled analytes within clumps. In addition, all analytes can be kept away from potentially adherent surfaces during dielectric separations so that sample recovery efficiency is improved. Nevertheless, the intrinsic dielectric properties of target particles, cells, or molecules, or of their dielectric labels may still not allow for sufficient discrimination between multiple target analytes in complex mixtures.
Furthermore, the existing dielectric methods having the most discrimination between different analytes (termed Dielectrophoretic Field-Flow Fractionation (DEP-FFF methods) exploit a balance between dielectrophoretic and sedimentation forces on analytes in the sample mixture. Such a balance can only be realized if there is a specific orientation of the apparatus with respect to a gravitational or centrifugal field. This precludes or limits the use of the methods for applications in microgravity environments such as space. The need to attain a balance between dielectrophoretic and sedimentation forces also places constraints on the relative densities of the suspending medium that carries the analyte mixture and the analytes to be separated. For example, the target analyte or, in the case of dielectric labeling, the analyte-label complex, must have a density that is slightly (typically 2–20%) higher than the suspending medium for effective DEP-FFF separation. Finally, because the sedimentation force acting on a target analyte or target analyte-label complex is usually small and uniform in space within the separation chamber, it typically takes many minutes for analytes or analyte-label complexes to reach positions in the dielectric separation apparatus where a balance of forces occurs.
Thus it is often necessary to allow a sample to sit for some “relaxation time” after it is introduced into a DEP-FFF separator to give analytes time to sediment before separation steps are initiated. Since this relaxation time is often comparable to the time taken to complete all of the rest of the separation steps combined, this step significantly slows the separation procedure and is inconvenient.